Alcohol-induced liver injury involves significant mitochondrial damage and up-regulation of inducible nitric oxide synthase (NOS2) leading to excessive generation of reactive oxygen species (ROS) and reactive nitrogen species affecting cell survival. Understanding the molecular mechanisms of pathological nitric oxide (NO) overproduction by NOS2 is of great relevance for potential therapeutic intervention. Our laboratory has used a combination of a cutting edge proteomics technique along with a Systems Biology approach to elucidate mitochondrial proteins involved in ALD that could impact NO synthesis. We have identified argininosuccinate synthase (ASS) as up-regulated by chronic alcohol feeding. Patients with alcoholic liver disease (ALD) or with hepatocellular carcinoma also showed increased hepatic ASS suggesting a potential link between ASS and ALD. ASS is an enzyme from the L-citrulline/NO cycle which could have a rate-limiting role for high-output NO synthesis via NOS2. Virtually nothing is known on how alcohol modulates ASS expression and how the L-arginine recycling pathway may impact NO generation and liver injury. We hypothesize that up-regulation of ASS by alcohol-derived species may increase the availability of intracellular substrate for NO synthesis by NOS2 contributing to the pathophysiology of ALD. We will test this hypothesis and study the mechanistic aspects involved in ASS induction and the biological relevance along the following Specific Aims: 1) To dissect whether the alcohol-mediated up-regulation of ASS plays a role in increased NO synthesis by NOS2 in hepatocytes, we will address: a) whether the up-regulation of ASS by alcohol increases intracellular L-arginine availability for NO synthesis by NOS2 in hepatocytes, and b) whether alcohol induces L-arginine influx playing a role in NO synthesis; 2) To identify the mechanism by which alcohol induces ASS, we will consider: a) if increased ROS act as sensors leading to up-regulation of ASS by alcohol, and b) if ASS undergoes S-nitrosylation to regulate NO production indicating a novel alcohol-related feedback mechanism whereby NO limits its own synthesis by governing substrate regeneration for high-output NO synthesis; and 3) To assess the biological relevance of the alcohol-mediated induction of ASS for NO synthesis in vivo, wild-type mice, Ass, and mice injected with either control EGFP-AAV8 or ASS-EGFP-AAV8 will be fed the control or the alcohol Lieber-DiCarli diets and liver function and biochemical measures of oxidative and nitrosative stress will be evaluated as a read-out for the contribution of ASS to liver injury. Such information could lead to positive pharmacological targeting to ameliorate alcohol hepatotoxicity.